Rotavirus (RV) causes diarrhea in infants worldwide with most deaths in Africa and Asia. Newly licensed oral RV vaccines have unproven or lower efficacies in infants in impoverished countries. Their high costs remain obstacles to universal adoption. In developing countries, widespread vitamin A deficiencies (VAD) compromise children's health, gut immune responses and vaccine effectiveness. WHO has linked vitamin A (VitA) supplementation in infants to immunization programs to reduce VAD and increase vaccine coverage. The impact of VitA supplementation on RV vaccines is unknown. Our innovative and novel strategy is to use supplemental VitA as an adjuvant with oral vaccines in neonates. New discoveries have revealed that VitA is a key regulator of intestinal immune responses and that VAD impairs gut immune responses. Gut dendritic cells (DCs) convert dietary VitA to retinoic acid (RA) and DCs and/or RA in concert with key signaling molecules (cytokines, inflammatory mediators), imprint gut homing (CCR9, a4[unreadable]7) on T and B cells, promote isotype switching to IgA and enhance IgA production. In infants and piglets, fecal or serum IgA antibodies or IgA antibody secreting cells in the gut correlate with protection against RV diarrhea. Gut DCs orchestrate critical upregulation of intestinal immune responses (via Teffector, B cells and mediators) to enteric pathogens or downregulation (via immunoregulatory T cells and mediators like TGF[unreadable]) to food antigens and commensals. We hypothesize that "danger signals" consisting of inflammatory or TLR signaling are essential to activate DCs to overcome intestinal downregulation and increase immune responses to oral vaccines. To test this, we will use attenuated RV that replicates in enterocytes, (increase proinflammatory, decrease TGF[unreadable] cytokines) as oral vaccine with VitA adjuvant to enhance gut IgA responses. Alternatively nonreplicating rotavirus-like-particle (VLP) vaccines may require TLR signals (polyinosine-polycytidylic acid synthetic dsRNA, TLR3 ligand) with VitA to mimic the effect of RV dsRNA. Using VitA as adjuvant, we will compare immune responses to live attenuated (Aim 1) with VLP (Aim 2) oral RV vaccines. Vaccine efficacy will be tested in VAD versus VitA replete neonatal gnotobiotic (Gn) pigs challenged with virulent human RV (Aim 3). Neonatal Gn piglets mimic infants in their gastrointestinal physiology, mucosal immune responses and susceptibility to human RV diarrhea. Pigs metabolize VitA like humans and VitA levels can be manipulated to mimic VAD in infants. To elucidate immune mechanisms, we will assess innate (DCs, NK/NKT cells, IFNa, inflammatory cytokines) and adaptive T (Teff/Treg ratio) and B cell (IgA) intestinal immune responses, including gut DC and cytokine profiles and their correlation with protection. Understanding the impact of VAD on gut immunity is critical to improve oral vaccine efficacy in VAD infants in developing countries. If successful, strategic implementation of supplemental VitA in infants (as recommended by WHO) with existing RV oral vaccines may be feasible and could provide more cost-effective vaccines (lower, fewer doses) in developing countries. An innovative outcome would be universal application of VitA adjuvants not only to RV vaccines, but also to other mucosal vaccines such as for HIV. RELEVANCE: Rotavirus (RV) causes 660,000 childhood deaths annually worldwide, with most (90%) in Africa and Asia. Although live oral rotavirus vaccines are licensed, their unproven or lower effectiveness in infants in impoverished countries, where they are most needed, and high costs remain obstacles to their universal adoption. In developing countries, widespread malnutrition with micronutrient deficiencies (vitamin A, etc) and multiple concurrent infections compromise children's health, gut immune responses and vaccine effectiveness. New discoveries have revealed that vitamin A is a key regulator of intestinal immune responses and that vitamin A deficiency impairs gut immune responses. We will test supplemental vitamin A as adjuvant with live or nonreplicating oral rotavirus vaccines in vitamin A deficient versus vitamin A replete neonatal gnotobiotic pigs. Our studies will elucidate the immune responses and correlates of protection to challenge with virulent human rotavirus. Neonatal gnotobiotic piglets will be used as a disease model because they are the only animal susceptible to human rotavirus diarrhea. Moreover they are similar to infants in anatomy, gastrointestinal physiology, nutrient requirements/metabolism, and development of mucosal immunity. Also swine metabolize retinol comparable to humans. Thus the effects of vitamin A on the porcine immune system most closely mimic that in humans, and piglets best mirror responses of infants to rotavirus diarrhea and vaccines. An understanding of impacts of vitamin A deficiency on gut immune responses is critical to improve oral vaccine efficacy in vitamin A compromised infants in developing countries. If successful, immediate implementation of supplemental vitamin A in infants (as recommended by WHO) with the existing RV vaccines may be feasible and could provide more cost-effective vaccines (lower, fewer doses) in developing countries. Vitamin A adjuvants may be universally applicable, not only to rotavirus vaccines, but also to other mucosal vaccines for humans such as HIV.